Genotypic and phylogenic analyses of cutaneous leishmaniasis in Al Ahsa, Eastern Saudi Arabia during the coronavirus disease 2019 pandemic: First cases of Leishmania tropica with the predominance of Leishmania major

During the coronavirus disease 2019 lockdown period, a surge in sandflies and cutaneous leishmaniasis (CL) cases was observed in Al-Ahsa, Saudi Arabia. Skin punch biopsies were obtained from 100 patients clinically diagnosed with CL in Al-Ahsa who had no travel history in the last 6 months. Impression smears were used following a three-step polymerase chain reaction (PCR) protocol using genus-specific primers targeting kDNA and ITS1. Leishmania speciation was determined by ITS1 PCR/nested PCR-restriction fragment length polymorphism and sequencing. A phylogenetic tree was constructed. The associated patient characteristics were analyzed. Using internal transcribed spacer one (ITS1)-PCR/nested PCR, 98 cases were considered true-positive CL. Leishmania major was the predominant species, and Leishmania tropica was identified in three cases. Microscopy had poor sensitivity and perfect specificity. Direct ITS1-PCR missed nine cases. Sex, residence, and treatment outcome were significantly associated with the occurrence of Leishmania; distribution of skin lesion(s) and treatment outcome were significantly associated with Leishmania genotype. This is the first time that L. tropica was identified as a cause of CL in human in Al-Ahsa, in addition to the predominant zoonotic species, L. major. We recommend using ITS1-nested PCR for negative cases by ITS1-PCR. Further exploration of Leishmania transmission dynamics in vectors and reservoir animals is essential for designing effective preventive measures.


Scientific Reports
| (2022) 12:10753 | https://doi.org/10.1038/s41598-022-14702-z www.nature.com/scientificreports/ ing tool, followed by the protocol published by Schönian et al. 29 which uses two genus-and species-specific PCRs ( Fig. 1). A total of 25 µL was used for all PCR amplifications. Each reaction had 12.5 uL of Master mix, one uL of each primer, 7.5 uL RNA-free water, and three uL of sample's DNA which was around 25 ng. Leishmania major MHOM/TM/82/Lev and Leishmania tropica MHOM/SU/80/K28 strains were used as positive control and RNAfree water were used as negative controls, respectively. PCR inhabitation control reactions were carried out to exclude false-negative results. All PCR reagents were purchased from Promega (USA). Primers were purchased from Macrogen (Seoul, South Korea). Table 1 shows the primers used and reaction cycling conditions applied for each PCR assay used in this study.
All PCR amplified products were analyzed by electrophoresis on 1% agarose at 120 V in 1 × Tris-acetateethylenediaminetetraacetic acid buffer and were visualized under ultraviolet light.
Genotyping of Leishmania species. Leishmania species were determined using ITS1 PCR/nPCR-RFLP and sequencing. All positive PCR products from direct ITS1 PCR and ITS1 nPCR were analyzed using restriction enzyme analysis for species identification.
Each PCR-amplified product (10 µL) was digested with 1 U HaeIII enzyme (MOLEQULE-ON, New Zealand) at 37 °C for 15 min, following the kit guidelines. The RFLP product was examined on MetaPhor (4%) agarose gel (Lonza, Switzerland) and visualized under ultraviolet light.
The ITS1 PCR/nPCR products were processed by the Macrogen Company (South Korea) for sequencing. Sequencing was conducted in both directions.
All obtained sequences were submitted to the GenBank database (accession numbers OK560721-OK560817). All sequences were compared to the reference ITS1 sequences published in GenBank using BLAST (http:// www. ncbi. nlm. nih. gov). DNA sequences were aligned and trimmed using the BioEdit software (version 7.2.5.0; http:// www. mbio. ncsu. edu/ BioEd it/ bioed it. html). Neighbor-joining analysis was used to generate the phylogenetic tree using MEGAX software 39 . Bootstrap analyses with 1000 replicates were performed to assess the robustness of the constructed phylogenetic tree.  Informed consent. The purpose of the present study was comprehensively explained to all of the participants and the children's parents/guardians, and the collection of specimens was completed after obtaining their informed consent.
Polymerase chain reaction assays. Using the reference standard, 98 of the 100 cases were considered true-positive CL cases, whereas two of the 100 cases were considered true-negative cases. Based on direct ITS1 PCR 89 of the 98 (90.8%, P < 0.001) cases were detected, it had a sensitivity of 90.8%, poor NPV (18.2%), and  Identification of Leishmania species using restriction fragment length polymorphism analysis. Identification of Leishmania species was performed by digesting PCR products for the 98 true-positive samples using HeaIII restriction enzyme to distinguish each sample's RFLP pattern (Fig. 3). The predominant species was L. major; it was identified in 95 (96.9%) of the total patients' sample. For the first time in Al-Ahsa, L. tropica was identified in three patients (3.1%) ( Table 4).
Sequencing analysis. Sequencing analysis confirmed RFLP results with 100% similarity of the study DNA sequences with the sequences previously submitted to the GenBank, as shown in the constructed phylogenetic tree (Fig. 4).

Sociodemographic and clinical data of study individuals and their association with the occurrence of cutaneous leishmaniasis and Leishmania species.
Most patients were men (90%) and lived in rural areas (98%). More than half (61%) of the patients with CL were aged 15-44 years. There was no travel history for the participants in the last 6 months prior to the presentation. Skin lesions in the patients with CL were distributed in different body areas, and 50% of the lesions were distributed in the legs. Most of the patients (76.5%) had skin lesions at one body site. The patient presented with    5). Ulcerative plaque lesions were the most common presentation (28%), followed by ulcerative lesion (19%), then nodular (17%), and plaque (13%) lesions. The patients were treated according to the Saudi Ministry of Health CL treatment guidelines 44 . The choice of therapy was decided according to the clinical presentation, stage, number, and sites of the CL skin lesions. Most of the patients (94/98, 96%) were treated with sodium stibogluconate either intramuscularly or intra-lesionally. Only four (4%) patients were treated with oral fluconazole. Complete resolution of lesions after the entire course    (Fig. 6). One patient required a second course of oral fluconazole for 4 weeks after the first course to achieve complete resolution. One patient had recurrence of the lesions after a full course of therapy (lesions appeared after 4 months of complete resolution). The sociodemographic and clinical data of the patients were analyzed for their association with the molecular diagnosis of Leishmania and Leishmania species using ITS1 PCR/nPCR-RFLP (Tables 5 and 6). Among the studied patient characteristics, only sex, residence, and treatment outcome were associated with the occurrence of Leishmania with statistical significance (P < 0.05). Distribution of skin lesion(s) and treatment outcome were statistically significantly associated with the genotype of Leishmania (P < 0.05).

Discussion
Molecular assays, such as PCR, are highly specific and sensitive for the diagnosis of infectious diseases, including CL. The molecular identification of Leishmania species was performed by following a modified protocol for genus-specific primers targeting kDNA as the first screening step 32 , followed by the use of two genus-and species-specific PCRs, ITS1 PCR and ITS1 nPCR, using the first ssu rRNA PCR (Fig. 1). This modification aimed to study the use of kDNA primer as a first-step screening tool to be implemented in routine diagnostic laboratories and thus overcome the possible false-negative results by Schönian et al. 29 . In our study, 98 of the 100 samples were considered true positive using our reference standard molecular method. Treatment was initiated by the clinical team if the sample was true positive.
The PCR of kDNA was positive in all samples, including two false-positive results. Clinical confirmation of false positivity was performed in the case without initiation of the CL treatment protocol. Both patients showed spontaneous resolution of the lesions after two weeks. kDNA PCR was able to detect as low as 10 -4 parasites in patient samples 45 . Compared to other DNA targets, the kDNA target has abundant copy numbers (almost 10,000 copies) that are 50-250-fold higher than those of other PCR targets 23 . Based on our findings, the use of kDNA as a screening tool is discouraged as it may lead to false-positive results and is an extra step, whereas using ITS1 nPCR for negative cases by ITS1 PCR allows for accurate diagnosis and speciation of Leishmania. Other studies have suggested the use of kDNA as a screening tool with excellent performance 32,46 . Further comprehensive studies using larger sample sizes are required to assess kDNA PCR as a first-step screening tool. Based on direct ITS1-PCR, 89 of the 98 (90.8%, P < 0.001) cases were detected with 100% specificity and 90.8% sensitivity. Most published studies have reported similar results, with a sensitivity ranging from 63.5% to 100% 23,32,46-49 and a specificity ranging from 93 to 100% 23,32,[47][48][49] . The different methods used for sample collection in published studies could explain the wide range of sensitivities. Studies using punch biopsy or aspirate showed higher performance. Direct ITS1-PCR PPV was 100%, whereas the NPV was poor (18.2%). The poor NPV value compared to other   23,32,48,49 can be explained by our inclusion criteria, which only recruited patients referred to the regional CL clinic with a presumptive diagnosis of CL. Implementing ITS1 nPCR for negative samples using ITS1 PCR as a reference standard enhanced the sensitivity and overcame the false-negative results. Sequencing analysis confirmed the ITS1 PCR/nPCR-RFLP results for Leishmania species identification. In the Al-Ahsa region, L. major was the predominant zoonotic species. Nevertheless, L. tropica was identified in three of the study participants. There was no travel history for any of the study individuals in the last six months prior to presentation. To the best of our knowledge, this is the first study to identify L. tropica in human CL cases in the Al-Ahsa region.
Previously, Al-Salem et al. 33 identified only L. major in clinical samples from the Al-Ahsa region. A similar species distribution was identified in other regions of Saudi Arabia. El-Beshbishy et al. 31,32 found that L. major and L. tropica were the CL species present both in field-caught sandflies 31 and in clinical samples 32 in Al-Madinah Al-Munawwarah, western province of Saudi Arabia. The predominant Leishmania species was L. major. Rasheed et al. 50 identified L. major, L. tropica, and a few cases of L. infantum/ Leishmania donovani in Qassim, central region of Saudi Arabia. In Asir and Jazan in southwestern Saudi Arabia, L. tropica is considered the predominant species 33 . Despite findings from all previous studies in Saudi Arabia, which exclusively reported the presence of the anthroponotic type of L. tropica, the zoonotic form of L. tropica was reported in Al-Ahsa in seven of the 175 samples collected from stray dogs by Alenezi et al. 51 . They only used two rounds of the less discriminative kDNA PCRs for genus and species identification to identify L. tropica in dogs, and they did not specify whether the samples had been collected from transported or local dogs.
In our study, the identification for the first time of L. tropica in human samples in the Al-Ahsa region can be explained by the migration of sandflies from other regions of Saudi Arabia during the COVID-19 pandemic lockdown that led to the cessation of vector-borne disease prevention programs and affected sandfly surveillance. The WHO and many experts have concluded that vector densities and health threats from vector-borne diseases can increase and even double as a result of restrictions in human mobility due to the COVID-19 lockdown, which may adversely affect the epidemiology of vector-borne diseases [52][53][54][55] .
More generally, the pattern of Leishmania species distribution in certain localities can easily change over time due to changes in the environment with rapid urbanization, vector movement, vector potency, vector control measures, animal transportation, and climate changes 56 . www.nature.com/scientificreports/ Despite the poor performance of microscopic examination as a diagnostic tool, it is still considered the gold standard for diagnosing CL in clinically diagnosed patients in many laboratories worldwide 23 . Microscopy was limited by poor sensitivity, with missed diagnosis in 65 (66.3%) patients. Other studies have reported similar low sensitivity (37-62.6%) of microscopy to detect Leishmania amastigotes in stained skin smears 23,32,57-60 . The poor performance of this tool is explained by the need for a high number of viable parasites in the sample to be visualized, the extreme demand for an expert microscopist to read the smears, and the flawed methods of sample collection. Therefore, a reliable and cost-effective diagnostic tool for the diagnosis of CL is essential.
Multiple studies have addressed the relationship between the clinical and epidemiological characteristics of CL cases and the occurrence of Leishmania and Leishmania species 33,[61][62][63][64][65] . In the current study, the area of residence of the study participants (P < 0.001) and the clinical outcome of treatment (P < 0.001) were significantly associated with PCR results, whereas family history (0.028), distribution of cutaneous lesions (0.038), and clinical outcome of treatment (P < 0.001) were significantly associated with ITS1-PCR RFLP results. In a previously published study in Saudi Arabia, multiple clinical factors, including lesion location, number, stage, and presence of satellite lesions, were significantly associated with species type 33 . El-Badry et al. 61 reported a statistically significant association between the age group of affected individuals and the ITS1-PCR results in Libya. Other studies have reported a significant relationship between molecular characterization and different clinical and epidemiological factors, such as CD4 count 62 , presence of hyrax in the villages, cases of CL in neighboring houses 63 , history of chronic disease and duration of lesions 64 and factors related to indoor dwelling environment 65 . These significant variables may also be predictors of the occurrence of Leishmania and Leishmania species in the region. The main limitation of our study is the lack of comprehensive clinical correlation between the species detected by the molecular techniques and the patients' clinical presentation and demographics. Further studies are needed to confirm our results.

Conclusion
To the best of our knowledge, this comprehensive molecular study fills a gap in the knowledge regarding the identification of the prevalent Leishmania species causing CL in the Al-Ahsa region. For the first time, our study detected human cases of anthroponotic L. tropica in Al-Ahsa. For accurate molecular diagnosis and speciation of Leishmania, we recommend using ITS1 nPCR for negative cases by ITS1 PCR. Further studies on vectors and animal reservoirs are essential to identify Leishmania transmission dynamics in the Al-Ahsa region. This crucial information can help guide the future planning of control methods to prevent the spread of CL in this region.

Data availability
The datasets generated and/or analyzed in the current study are available in the GenBank database (accession numbers: OK560721-OK560817).